2.4.4. Antimicrobial Activity

For the food to be shielded from microbial development and kept fresh for a long time, the active food packaging sheet must have strong antimicrobial activity [48]. Table 5 displays the antibacterial activity of four Gram-positive and Gram-negative bacteria against CH edible gel films combined with EPPE at various percentages.

**Table 5.** Antibacterial activity of chitosan films modified with different percentages of EPPE.


Data are presented as mean ± SD. Means with different superscripts (a–d) in lowercase letters in a column are significantly different at *p* < 0.05. NA means non-active.

As shown in Table 5, the control films (Ch-0%-EPPE) were ineffective against either of the four bacterial strains, but with the addition of EPPE, all the tested films showed antimicrobial activity on the contact area beneath the film discs. Our results were in accordance with those of Wang et al. [49], who found no significant inhibitory zone for the pure CH-gel film towards both Gram-positive and Gram-negative bacteria. This impact of CH may be connected to the fact that in the agar diffusion test method, chitosan does not diffuse across the neighbouring agar medium, meaning that only bacteria in direct contact with the active discs of CH are inhibited. The positively charged amino groups of the CH molecules, which may interact with the anionic groups on the microbial cell membrane, are also necessary for the antibacterial efficacy of chitosan [49]. In such instances, CH has been shown to exhibit intrinsic antibacterial activity against both Gram-positive and Gram-negative bacteria [22]. The Ch-EPPE films, in general, showed inhibitory effects (*p* < 0.05) on both Gram-positive and Gram-negative bacteria, and inhibition zones grew larger as the EPPE percentage rose in the film. According to the data reported in the same table, *Salmonella typhimurium* is the most sensitive to the films, followed by *E. coli, Pseudomonas aeruginosa,* and *Bacillus subtilis*. According to this study, EPPE worked better against Gram-positive bacteria than it did against Gram-negative bacteria. This could be due to variations in cell wall structure, as the cell walls of Gram-negative bacteria have lipopolysaccharides, which may prevent active constituents from entering the cytoplasmic membrane [50,51]. The primary location of contact for polyphenols with bacteria is the outer cell membrane [52]. In the polyphenols, the hydroxyl groups, conjugated double bonds, and galloyl groups are the active groups in charge of this interaction. The bacteria may die if the membrane, which protects the cell's integrity, is damaged as a result of this contact. Gram-positive bacteria than it did against Gram-negative bacteria. This could be due to variations in cell wall structure, as the cell walls of Gram-negative bacteria have lipopolysaccharides, which may prevent active constituents from entering the cytoplasmic membrane [50,51]. The primary location of contact for polyphenols with bacteria is the outer cell membrane [52]. In the polyphenols, the hydroxyl groups, conjugated double bonds, and galloyl groups are the active groups in charge of this interaction. The bacteria may die if the membrane, which protects the cell's integrity, is damaged as a result of this contact. *2.5. Films SEM Photographs*  Figure 2A–D depict the results from a prepared film SEM study. The control gel film

*Gels* **2023**, *9*, x FOR PEER REVIEW 8 of 16

column are significantly different at *p* < 0.05. NA means non-active.

Data are presented as mean ± SD. Means with different superscripts (a–d) in lowercase letters in a

of the four bacterial strains, but with the addition of EPPE, all the tested films showed antimicrobial activity on the contact area beneath the film discs. Our results were in accordance with those of Wang et al. [49], who found no significant inhibitory zone for the pure CH-gel film towards both Gram-positive and Gram-negative bacteria. This impact of CH may be connected to the fact that in the agar diffusion test method, chitosan does not diffuse across the neighbouring agar medium, meaning that only bacteria in direct contact with the active discs of CH are inhibited. The positively charged amino groups of the CH molecules, which may interact with the anionic groups on the microbial cell membrane, are also necessary for the antibacterial efficacy of chitosan [49]. In such instances, CH has been shown to exhibit intrinsic antibacterial activity against both Gram-positive and Gram-negative bacteria [22]. The Ch-EPPE films, in general, showed inhibitory effects (*p* < 0.05) on both Gram-positive and Gram-negative bacteria, and inhibition zones grew larger as the EPPE percentage rose in the film. According to the data reported in the same table, *Salmonella typhimurium* is the most sensitive to the films, followed by *E. coli, Pseudomonas aeruginosa,* and *Bacillus subtilis*. According to this study, EPPE worked better against

As shown in Table 5, the control films (Ch-0%-EPPE) were ineffective against either

### *2.5. Films SEM Photographs* (ch-0%-EPPE) has a smooth, uniform surface. There were no impurities, delamination, or

Figure 2A–D depict the results from a prepared film SEM study. The control gel film (ch-0%-EPPE) has a smooth, uniform surface. There were no impurities, delamination, or precipitates (Figure 2A). The studied films' surface morphology was unaffected by the addition of 1% EPPE, it appears (Figure 2B). Small agglomerates were seen when CH was added with 3% EPPE (Figure 2C). A more heterogeneous surface was produced as a result of the structure being disturbed by the rise in EPPE concentration from 3% to 5%, which led to the appearance of more and more white patches (Figure 2D). This could be because EPPE contains hydrophilic polyphenolic components. precipitates (Figure 2A). The studied films' surface morphology was unaffected by the addition of 1% EPPE, it appears (Figure 2B). Small agglomerates were seen when CH was added with 3% EPPE (Figure 2C). A more heterogeneous surface was produced as a result of the structure being disturbed by the rise in EPPE concentration from 3% to 5%, which led to the appearance of more and more white patches (Figure 2D). This could be because EPPE contains hydrophilic polyphenolic components.

**Figure 2.** Active films' SEM images, where (**A**) Control (Ch-0% EPPE), (**B**) Ch-1% EPPE, (**C**) Ch-3% EPPE, and (**D**) Ch-5%EPPE. **Figure 2.** Active films' SEM images, where (**A**) Control (Ch-0% EPPE), (**B**) Ch-1% EPPE, (**C**) Ch-3% EPPE, and (**D**) Ch-5%EPPE.
